药物诱导MDA-MB-435细胞表达ERα及其对内分泌治疗的敏感性

Expression of ERαin chemically induced MDA-MB-435 cells and its responsiveness to endocrine therapy

目的通过药物诱导雌激素受体(ER)阴性的乳腺癌细胞株MDA-MB-435恢复ER的表达,探讨其对内分泌治疗的敏感性。方法采用HDAC抑制剂古曲抑菌素(TSA)联合DNMT抑制剂5-AZA-CdR(AZA)作用于MDA-MB-435细胞;以RT-PCR方法检测细胞ERα及其下游基因产物孕激素受体(PR)、雌激素调节蛋白(pS2)的mRNA水平;以水溶性四氮唑盐-8(WST-8)方法检测诱导前后细胞增殖水平。将药物诱导后的MDA-MB-435细胞接种裸小鼠,观察其在体内增殖能力的改变。结果药物诱导后,MDA-MB-435细胞恢复ERα表达,ERα下游基因产物PR和pS2的表达增加。2．5μmol／L AZA和100 ng／ml TSA诱导MDA-MB-435细胞后,其ERα表达最强。诱导后的MDA- MB-435细胞在不同浓度的雌激素中表现出不同的增殖能力。与未经诱导的细胞相比,诱导后的MDA-MB-435细胞增殖能力下降(P<0．01);经诱导的细胞联合4-羟基三苯氧胺后,其增殖能力进一步下降(P<0．01);而单用4-羟基三苯氧胺不能抑制未经诱导的细胞的增殖(P>0．05)。在裸鼠体内,诱导处理后的MDA-MB-435移植瘤较未经处理的细胞增殖能力下降(P<0．01)。去除雌激素后,诱导后肿瘤细胞在体内增殖能力进一步受到抑制(P<0．01)。结论通过TSA和AZA联合诱导作用,MDA-MB-435细胞株中沉默的ER恢复表达ERα,并且该ERα是具有功能的。经过诱导的细胞株在体内及体外对雌激素均恢复反应。联合应用HDAC抑制剂以及DNMT1抑制剂可以恢复乳腺癌细胞MDA-MB-435对内分泌治疗的敏感性,这为ER阴性的乳腺癌患者治疗开辟一条新的思路,具有重要的临床意义。

Objective To investigate the expression of ERα in chemically induced, ER α-negative human breast cancer MDA-MB-435 cells and its restoration of the responsiveness to endocrine therapy. Methods MDA-MB-435 cells were treated with HDAC inhibitor trichostatin A（TSA）and DNMT1 inhibitor 5-AZA-CdR （AZA）. The mRNA level of ERα, PR and PS2 in treated MDA-MB-435 cells was detected by RT-PCR. The WST-8 （water-soluble tetrazolium salt-8） method was used to analyze the proliferation rate of the cells. Xenograft in female nude mice was used to further explore the change of proliferation rate of treated MDA-MB-435 cells in vivo. Results After treatment with AZA and TSA, mRNA expression of ERα, PR and pS2 was up-regulated in MDA-MB-435 cells. The mRNA level of ERα was the hightest when MDA-MB- 435 cells were treated with 2.5 μmol/L AZA and 100 ng/ml TSA. The treated MDA-MB-435 cells showed different proliferation rate in various media containing different concentration of estredial. The MDA-MB-435 cells showed down-regulated proliferation rate after treatment with the combination of 2.5 μmol/L AZA and 100 ng/ml TSA, and 4-OH tamoxifen could suppress the growth rate of the induced MD-MBA-435 cells but not the untreated cells. The treated MDA-MB-435 cells showed slower proliferation rate than that of untreated cells in vivo （P 〈0.01 ）, and the proliferation rate of the treated MDA-MB-435 cells became lower when the nude mice were deprived of estrogen by castration （ P 〈 0.01 ）. Conclusion After treatment with TSA and AZA, ER α-negative MDA-MB-435 cells can express functional ERa and regain responsiveness to estrogen both in vitro and in vivo. HDAC inhibitor and DNMT1 inhibitor may play an important role in restoration of sensitivity of ER α-negative breast cancers to endocrine therapy.